RESUMEN
Uteroglobin, a steroid-inducible, cytokine-like, secreted protein with immunomodulatory properties, has been reported to bind progesterone, polychlorinated biphenyls (PCB), and retinol. Structural studies may delineate whether binding of ligands is a likely physiological function of human uteroglobin (hUG). We report a refined crystal structure of uncomplexed recombinant hUG (rhUG) at 2.5-A resolution and the results of our molecular modeling studies of ligand binding to the central hydrophobic cavity of rhUG. The crystal structure of rhUG is very similar to that of reported crystal structures of uteroglobins. Using molecular modeling techniques, the three ligands--PCB, progesterone, and retinol--were docked into the hydrophobic cavity of the dimer structure of rhUG. We undocked the progesterone ligand by pulling the ligand from the cavity into the solvent. From our modeling and undocking studies of progesterone, it is clear that these types of hydrophobic ligands could slip into the cavity between helix-3 and helix-3' of the dimer instead of between helix-1 and helix-4 of the monomer, as proposed earlier. Our results suggest that at least one of the physiological functions of UG is to bind to hydrophobic ligands, such as progesterone and retinol.
Asunto(s)
Modelos Moleculares , Unión Proteica/fisiología , Estructura Terciaria de Proteína/fisiología , Proteínas Recombinantes/química , Uteroglobina/química , Animales , Cristalografía por Rayos X/métodos , Humanos , LigandosRESUMEN
Uteroglobin (UG) is a multifunctional, secreted protein with anti-inflammatory and antichemotactic properties. While its anti-inflammatory effects, in part, stem from the inhibition of soluble phospholipase A2 (sPLA2) activity, the mechanism(s) of its antichemotactic effects is not clearly understood. Although specific binding of UG on microsomal and plasma membranes has been reported recently, how this binding affects cellular function is not clear. Here, we report that recombinant human UG (hUG) binds to both normal and cancer cells with high affinity (20-35 nM, respectively) and specificity. Affinity cross-linking studies revealed that 125I-hUG binds to the NIH 3T3 cell surface with two proteins of apparent molecular masses of 190 and 49 kDa, respectively. UG affinity chromatography yielded similar results. While both the 190- and 49-kDa proteins were expressed in the heart, liver, and spleen, the lung and trachea expressed only the 190-kDa protein. Some cancer cells (e.g., mastocytoma, sarcoma, and lymphoma) expressed both the 190- and 49-kDa proteins. Further, using functional assays, we found that UG dramatically suppressed the motility and extracellular matrix invasion of both NIH 3T3 and some cancer cells. In order to further characterize the anti-ECM-invasive properties of UG, we induced expression of hUG into cancer cell lines derived from organs that, under physiological circumstances, secrete UG at a high level. Interestingly, it has been reported that a high percentage of the adenocarcinomas arising from the same organs fail to express UG. Our results on induced hUG expression in these cells show that inhibition of motility and ECM invasion requires the expression of both UG and its binding proteins. Taken together, our data define receptor-mediated functions of UG in which this protein regulates vital cellular functions by both autocrine and paracrine pathways.
Asunto(s)
Proteínas Portadoras/metabolismo , Movimiento Celular/genética , Invasividad Neoplásica/genética , Unión Proteica/genética , Receptores de Superficie Celular/metabolismo , Células Tumorales Cultivadas/metabolismo , Uteroglobina/metabolismo , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/farmacología , Movimiento Celular/efectos de los fármacos , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/metabolismo , Humanos , Ratones , Invasividad Neoplásica/patología , Unión Proteica/efectos de los fármacos , Ensayo de Unión Radioligante/estadística & datos numéricos , Receptores de Superficie Celular/efectos de los fármacos , Receptores de Superficie Celular/aislamiento & purificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/patología , Uteroglobina/genética , Uteroglobina/farmacologíaRESUMEN
Blastokinin or uteroglobin (UG) is a steroid-inducible, evolutionarily conserved, multifunctional protein secreted by the mucosal epithelial of virtually all mammals. It is present in the blood and in other body fluids including urine. An antigen immunoreactive to UG antibody is also detectable in the mucosal epithelia of all vertebrates. UG-binding proteins (putative receptor), expressed on several normal and cancer cell types, have been characterized. The human UG gene is mapped to chromosome 11q12.2 13.1, a region that is frequently rearranged or deleted in many cancers. The generation of UG knockout mice revealed that disruption of this gene causes: (i) severe renal disease due to an abnormal deposition of fibronectin and collagen in the glomeruli; (ii) predisposition to a high incidence of malignancies; and (iii) a lack of polychlorinated biphenyl binding and increased oxygen toxicity in the lungs. The mechanism(s) of UG action is likely to be even more complex as it also functions via a putative receptor-mediated pathway that has not yet been clearly defined. Molecular characterization of the UG receptor and signal transduction via this receptor pathway may show that this protein belongs to a novel cytokine/chemokine family.
Asunto(s)
Citocinas/fisiología , Uteroglobina/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Citocinas/química , Citocinas/genética , ADN Complementario/genética , Regulación de la Expresión Génica , Genes Supresores de Tumor , Humanos , Ratones , Ratones Noqueados , Ratones Transgénicos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Receptores de Citocinas/fisiología , Homología de Secuencia de Aminoácido , Uteroglobina/química , Uteroglobina/genéticaRESUMEN
Uteroglobin (UG) is a multifunctional, secreted protein that has receptor-mediated functions. The human UG (hUG) gene is mapped to chromosome 11q12.2-13.1, a region frequently rearranged or deleted in many cancers. Although high levels of hUG expression are characteristic of the mucosal epithelia of many organs, hUG expression is either drastically reduced or totally absent in adenocarcinomas and in viral-transformed epithelial cells derived from the same organs. In agreement with these findings, in an ongoing study to evaluate the effects of aging on UG-knockout mice, 16/16 animals developed malignant tumors, whereas the wild-type littermates (n = 25) remained apparently healthy even after 11/2 years. In the present investigation, we sought to determine the effects of induced-expression of hUG in human cancer cells by transfecting several cell lines derived from adenocarcinomas of various organs with an hUG-cDNA construct. We demonstrate that induced hUG expression reverses at least two of the most important characteristics of the transformed phenotype (i.e., anchorage-independent growth on soft agar and extracellular matrix invasion) of only those cancer cells that also express the hUG receptor. Similarly, treatment of the nontransfected, receptor-positive adenocarcinoma cells with purified recombinant hUG yielded identical results. Taken together, these data define receptor-mediated, autocrine and paracrine pathways through which hUG reverses the transformed phenotype of cancer cells and consequently, may have tumor suppressor-like effects.
Asunto(s)
Adenocarcinoma/genética , Adenocarcinoma/patología , Transformación Celular Neoplásica , Cromosomas Humanos Par 11 , Uteroglobina/genética , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Mapeo Cromosómico , Femenino , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Masculino , Ratones , Ratones Noqueados , Invasividad Neoplásica , Neoplasias Experimentales/genética , Fenotipo , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética , Transfección , Células Tumorales Cultivadas , Neoplasias Uterinas/genética , Neoplasias Uterinas/patología , Uteroglobina/deficiencia , Uteroglobina/fisiologíaRESUMEN
Uteroglobin (UG) is a steroid-inducible, multifunctional, secreted protein with antiinflammatory and antichemotactic properties. Recently, we have reported a high affinity UG-binding protein (putative receptor), on several cell types, with an apparent molecular mass of 190 kDa (Kundu, G. C., Mantile, G., Miele, L., Cordella-Miele, E., and Mukherjee, A. B. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 2915-2919). Since UG is a homodimer in which the 70 amino acid subunits are connected by two disulfide bonds, we sought to determine whether UG monomers also interact with the 190-kDa UG-binding protein and if so, whether it has the same biological activity as the dimer. Surprisingly, we discovered that in addition to the 190-kDa species, another protein, with an apparent molecular mass of 49 kDa, binds reduced UG with high affinity and specificity. Both 49- and 190-kDa proteins are readily detectable on nontransformed NIH 3T3 and some murine cancer cells (e. g. mastocytoma, sarcoma, and lymphoma), while lacking on others (e.g. fibrosarcoma). Most interestingly, pretreatment of the cells, which express the binding proteins, with reduced UG dramatically suppresses extracellular matrix (ECM) invasion, when such treatment had no effect on fibrosarcoma cells that lack the UG-binding proteins. Tissue-specific expression studies confirmed that while both 190- and 49-kDa UG-binding proteins are present in bovine heart, spleen, and the liver, only the 190-kDa protein is detectable in the trachea and in the lung. Neither the 190-kDa nor the 49-kDa protein was detectable in the aorta. Purification of these binding proteins from bovine spleen by UG-affinity chromatography and analysis by SDS-polyacrylamide gel electrophoresis followed by silver staining identified two protein bands with apparent molecular masses of 40 and 180 kDa, respectively. Treatment of the NIH 3T3 cells with specific cytokines (i.e. interleukin-6) and other agonists (i.e. lipopolysaccharide) caused a substantially increased level of 125I-UG binding but the same cells, when treated with platelet-derived growth factor, tumor necrosis factor-alpha, interferon-gamma, and phorbol 12-myristate 13-acetate, did not alter the UG binding. Taken together, these findings raise the possibility that UG, through its binding proteins, plays critical roles in the regulation of cellular motility and ECM invasion.